title: SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo.
abstract: Successful implantation and long-term survival of engineered tissue grafts hinges on adequate vascularization of the implant. Endothelial cells are essential for patterning vascular structures, but they require supportive mural cells such as pericytes/mesenchymal stem cells (MSCs) to generate stable, functional blood vessels. While there is evidence that the angiogenic effect of MSCs is mediated via the secretion of paracrine signals, the identity of these signals is unknown. By utilizing two functionally distinct human MSC clones, we found that so-called "pericytic" MSCs secrete the pro-angiogenic vascular guidance molecule SLIT3, which guides vascular development by directing ROBO4-positive endothelial cells to form networks in engineered tissue. In contrast, "non-pericytic" MSCs exhibit reduced activation of the SLIT3/ROBO4 pathway and do not support vascular networks. Using live cell imaging of organizing 3D vascular networks, we show that siRNA knockdown of SLIT3 in MSCs leads to disorganized clustering of ECs. Knockdown of its receptor ROBO4 in ECs abolishes the generation of functional human blood vessels in an in vivo xenogenic implant. These data suggest that the SLIT3/ROBO4 pathway is required for MSC-guided vascularization in engineered tissues. Heterogeneity of SLIT3 expression may underlie the variable clinical success of MSCs for tissue repair applications.
authors.affiliation: Section of Cardiology, Department of Medicine, University of Chicago, Chicago, IL.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Paul
authors.affiliation: Department of Pathology, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA.
authors.forename: Kareen L K
authors.initials: KLK
authors.lastname: Coulombe
authors.affiliation: Research Resources Center, University of Illinois at Chicago, Chicago, IL.|Department of Pharmacology, University of Illinois at Chicago, Chicago, IL.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Toth
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL.|Section of Cardiology, Department of Medicine, University of Illinois at Chicago, Chicago, IL.
authors.forename: Yanmin
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL.
authors.forename: Glenn
authors.initials: G
authors.lastname: Marsboom
authors.affiliation: Integrated Microscopy Core Facility, University of Chicago, Chicago, IL.
authors.forename: Vytas P
authors.initials: VP
authors.lastname: Bindokas
authors.affiliation: Department of Pathology, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA.
authors.forename: David W
authors.initials: DW
authors.lastname: Smith
authors.affiliation: Department of Pathology, Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA.|Department of Bioengineering, University of Washington, Seattle, WA.|Department of Medicine/Cardiology, University of Washington, Seattle, WA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Murry
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL.|Section of Cardiology, Department of Medicine, University of Illinois at Chicago, Chicago, IL.|University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, IL.
authors.forename: Jalees
authors.initials: J
authors.lastname: Rehman
mesh_terms: D000818:Animals; D002450:Cell Communication; D002465:Cell Movement; D016000:Cluster Analysis; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018919:Neovascularization, Physiologic / Q000235:genetics*; D020286:Pericytes / Q000166:cytology / Q000378:metabolism; D010641:Phenotype; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D015533:Transcriptional Activation; D000097905:Roundabout Proteins
chemical_list: D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins; C498990:SLIT3 protein, human
keywords: Angiogenesis; DAPT; EC; HAEC; HUVEC; MSC; MSC27a; MSC5; Mesenchymal stem cells; N-[(3,5-Difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethyletheyl ester; Revascularization; SLIT–ROBO signaling; VEGF; Vascular tissue engineering; endothelial cell; human MSC clonal line HS-27a; human MSC clonal line HS-5; human aortic endothelial cell; human umbilical vein endothelial cell; mesenchymal stem cell or marrow stromal cell; vascular endothelial growth factor